Use of tri-methylated-β-cyclodextrin and sub-2 μm silica particles for enantiomers separation by nano-liquid chromatography

• Nano-Liquid chromatography was used for the chiral separation of some drugs. • C18 SP combined with a methylated-β-cyclodextrin in the mobile phase was successful. • Chiral separation was influenced by the organic solvent in the mobile phase. • Nano-LC can be considered a good green analytical technique. A short capillary column packed with RP18 sub-2 µm particles was investigated for enantioseparation of selected racemic acidic compounds such as nonsteroidal anti-inflammatory drugs (carprofen, cicloprofen, fenoprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, naproxen, and suprofen) using a lab-made nano-liquid chromatography (nano-LC) system at conventional pressure conditions. The chiral separations were achieved by adding to the mobile phase heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) as the chiral additive. A comparative study with a 3 µm silica-based RP18 stationary phase highlights the crucial role of the stationary phase type. Despite similar or slightly higher enantioresolution factors, the most significant effects were the shorter analysis times (up to half) and higher theoretical plate numbers (estimated between 45,000 and 75,000 N/m) resulting from the reduced particle size of the stationary phase. This enabled a comprehensive organic solvent and aqueous content study, revealing valuable chromatographic insights. At a flow rate of approximately 400 nL/min, the most favourable mobile phase for enantiodiscrimination was achieved using 30 mM TM-β-CD in MeOH with a buffer pH 3.0. In line with the principles of Green Analytical Chemistry (GAC) and adhering to the White Analytical Chemistry principles, various tools were employed to assess the environmental impact of the method. These included the Analytical Eco-Scale, the Analytical GREEnness metric approach, and the Blue Applicability Grade Index that evaluated the miniaturized analytical methodology proposed aligned with the principles of Green Analytical Chemistry.